Biologically active methylene blue derivatives

ABSTRACT

The present invention relates to a pharmaceutical composition which contains a compound of formula 
                         
in which R 1 , R 2 , R 3  and R 4  are each n-butyl, where X P−  is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant.

This application is a divisional of U.S. application Ser. No.12/141,747, filed Jun. 18, 2008, which is a continuation of U.S.application Ser. No. 12/078,805, filed Apr. 4, 2008, which is acontinuation of U.S. application Ser. No. 10/723,420, filed Nov. 26,2003, now U.S. Pat. No. 7,371,744, which is a continuation-in-part ofPCT/GB02/02278, filed May 30, 2002, which claims priority from UKApplication No. 0113121.8, filed May 30, 2001 and UK Application No.0123945.8, filed Oct. 5, 2001, the entire contents of these applicationsbeing incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to biologically active photosensitisers theirconjugates, composites and compositions which are stronglyphotocytotoxic and have application in the areas of photodynamic therapy(PDT), as well as for the treatment, diagnosis and detection of medicalconditions and related uses in photochemical internalisation, in theproduction of cancer vaccines, in the treatment and prevention ofmicrobial infections and in photodisinfection or photosterilisation.

BACKGROUND TO THE INVENTION

It is known that certain organic compounds (“photosensitisers”) caninduce cell death by absorption of light in the presence of oxygen. Thecytotoxic effect involves Type I and/or Type II photooxidation. Suchphotosensitisers find use in the treatment of cancer and other diseasesor infections with light (photodynamic therapy) and in the sterilisation(including disinfection) of surfaces and fluids by the light-induceddestruction of microbes. In this context, the term sterilisation istaken to mean the reduction or elimination of microbes in a particularsituation. For prevention of wound infections sterilisation means asignificant reduction in bacterial load on, in or around a wound sitewhich helps to promote efficient wound healing or which minimises thechance that wound infection will occur.

It is also known that certain coloured phenothiazinium compounds, (e.g.methylene blue) can take part in Type I and Type II photooxidationprocesses, but compounds of this type thus far have proved unsuitable orof low efficacy as sensitisers for photodynamic therapy, or have shownlow photochemical antimicrobial activity.

For application in PDT, a good sensitiser must have at least some andpreferably all of the following properties. Most importantly, it shouldcause the destruction of target cells (for example tumour cells orbacterial cells) efficiently on exposure to light (preferablywavelengths ca. 600-800 nm). The PDT treatment using the photosensitisershould show a high degree of selectivity between target and normaltissues. The sensitiser should have relatively little dark toxicity andit should cause little or no skin photosensitivity in the patient. Thesensitiser should have short drug to light intervals for patient andhospital convenience and to minimise treatment costs.

For applications in photosterilisation, a good sensitiser must show astrong phototoxic effect in a wide range of microrganisms, ideally usingambient light, and should not photobleach readily.

In oncology, several different types of photosensitiser have been usedto treat both solid tumours and thin tumours of hollow organs such asthe oesophagus and bladder. However, the use of these photosensitisershas been restricted partly because of lack of selectivity between tumourand healthy tissue and partly because of the prolonged skinphotosensitivity which can be caused. There is a need for newphotosensitisers which cause little or no skin photosensitivity andwhich selectively destroy tumour cells.

Although PDT has previously been used in the treatment of tumours, ithas not yet been used clinically against infections caused by bacteriaand other microorganisms. The use of antibiotics to treat bacterialinfections is becoming challenging due to the increasing resistance ofmany bacterial species to commonly used antibiotics, such astetracyclines and beta-lactams. Hospital-acquired antibiotic resistantinfections such as MRSA are especially problematic. Photodynamicantibacterial treatment is a promising alternative to antibiotics forlocal treatment.

When developing antibacterial agents a major problem which must beovercome is the uptake of the drug into the bacterial cell. Gramnegative and Gram positive bacteria differ in the composition of theirouter surface and respond differently to antimicrobial agents,especially in terms of uptake. Due to the high negatively chargedsurface of Gram negative bacteria they are relatively impermeable toneutral or anionic drugs, including most commonly used photosensitisers.Development of antimicrobial photosensitisers which are effectiveagainst Gram negative bacteria, as well as Gram positive bacteria wouldbe highly beneficial to replace commonly used antibiotics and drugswhich are becoming increasingly ineffective due to resistance. A numberof different types of photosensitiser have been investigated inbacteria. These include phenothiazinium compounds, phthalocyanines,chlorins and naturally occurring photosensitisers. For uptake into Gramnegative bacteria, it is accepted that the cationic derivatives are themost effective.

Phenothiazinium compounds are blue dyes with maximum absorption atwavelengths between 600-700 nm. They have been studied for theirnon-photodynamic antibacterial properties but few apart from methyleneblue and toluidine blue have been investigated photodynamically.

Wainwright et al (1998) investigated the effect of a series ofphenothiazinium methylene blue derivatives in tumour cell lines andbacteria. New methylene blue (NMB) and di methyl methylene blue (DMMB)were effective at inactivating MRSA and were shown to be more effectivephotosensitisers than methylene blue when acting on pigmented melanomacell lines. Wagner et al (1998) studied these dyes and in addition ahydrophobic derivative for the inactivation of enveloped viruses.

The precise mode of antibacterial action of methylene blue is unknown,but one hypothesis is that because of its stereochemistry it canintercalate into DNA, and that photodynamic action causes DNA damage.Methylene blue itself has been shown to be ineffective as an anti-tumouragent. In addition, methylene blue is known to be susceptible tophotobleaching, which could be a serious disadvantage in someapplications. Because of the recognised limitations of methylene blue,both anti-tumour PDT and antimicrobial PDT would benefit fromdevelopment of new phenothiazinium-based photosensitisers.

STATEMENTS OF THE INVENTION

According the present invention there is provided a phenothiaziniumcompound of Formula (I):

wherein:A and B each independently is

in whichR′ and R″ each independently is an optionally substituted linear,branched or cyclic hydrocarbon group, or R′ and R″ together with the Natom to which they are attached form an optionally substituted 5-, 6- or7-membered ring;X^(P−) is a counteranion and P is 1, 2 or 3;except for the compounds in which A and B are both —N(CH₃)₂, or—N(CH₂CH₃)₂ for use in a treatment that requires removal, deactivationor killing of unwanted tissues or cells.

The linear and branched chain hydrocarbon groups represented by R′ andR″ contain from 1 to 12 carbon atoms, preferably from 1 to 10, morepreferably from 2 to 8, and especially from 2 to 6 carbon atoms. Theselinear and branched chain hydrocarbon groups may include one or moreunsaturated links, for example one or more alkene groups, and may beoptionally substituted by a group selected from H, F, Cl, Br, I, —OH,—OC₁₋₆-alkyl, —CN, —OCOC₁₋₆-alkyl or aryl. These linear and branchedchain hydrocarbon groups are preferably unsubstituted and are preferablysaturated hydrocarbon groups.

The cyclic hydrocarbon groups represented by R′ and R″ contain from 3 to8 carbon atoms, preferably from 4 to 6 carbon atoms and more preferably6 carbon atoms. These cyclic hydrocarbon groups may include one or moreunsaturated links, they may be optionally substituted and may optionallyinclude a heteroatom such as nitrogen.

Where R′ and R″ together with the N atom to which they are attached forman optionally substituted 5-, 6- or 7-membered ring the ring may containother heteroatoms and may be optionally substituted. The heteroatoms arepreferably selected from N, O or S. The heteroatoms may be substitutedby O, H or C₁₋₆-alkyl which is optionally substituted by —OH or —COCH₃,preferred substituted heteroatoms are selected from SO₂, NH, NCH₃,NC₂H₅, NCH₂CH₂OH and NCOCH₃. The optional ring substituents may beselected from —C₁₋₆-alkyl, —OH, —OC₁₋₆-alkyl, —OCOC₁₋₆-alkyl.

The counteranion represented by X^(P−) may be an organic or inorganiccounteranion and is preferably selected from F⁻, Br⁻, Cl⁻, I⁻, NO₃ ⁻,SCN⁻, ClO₃ ⁻, ClO₄ ⁻, IO₃ ⁻, BF₄ ⁻, HSO₄ ⁻, H₂PO₄ ⁻, CH₃SO₄ ⁻, N₃ ⁻, SO₄²⁻, HPO₄ ²⁻, PO₄ ³⁻, acetate, lactate, citrate, tartrate, glycolate,glycerate, glutamate, β-hydroxyglutamate, glucoaronate, gluconate,malate and aspartate.

In a preferred embodiment in the compounds of Formula I A and B eachindependently is selected from

in which Z is CH₂, CH₂—C₁₋₆-alkyl, O, S, SO₂, NH, NCH₃, NC₂H₅,NCH₂CH₂OH, or NCOCH₃ and R¹ and R² each independently is linear orbranched —C_(n)H_(2n)Y, where n is 1-10, preferably 1-8 and morepreferably 1-6, Y is H, F, Cl, Br, I, —OH, —OCH₃, —OC₂H₅, —OC₃H₇, —CN,—OCOCH₃ or phenyl, or R¹ and R² each independently is cyclohexyl, and inwhich each of the 5-, 6- or 7-membered rings above may carry one or more—C₁₋₆-alkyl, preferably —C₁₋₄-alkyl substituents and where X^(P−) is acounteranion and P is 1, 2 or 3.

In a further preferred embodiment R¹ and R² each independently is alinear or branched unsubstituted C₁₋₁₂ alkyl group or a group—C_(n)H_(2n)Y, where n is 1-6, Y is H, F, Cl, Br, I, —OH, —OCH₃, —OC₂H₅,—OC₃H₇, —CN or —OCOCH₃, or R¹ and R² each independently is cyclohexyl,or A and B each independently is selected from pyrrolidino orpiperidino. More preferably Y is H, F, Cl, Br, I, —OCH₃, —OC₂H₅, —OC₃H₇,—CN or —OCOCH₃,

Preferably the counteranion is selected from the group comprising Cl⁻,Br⁻, I⁻¹, F⁻, NO₃ ⁻, HSO₄ ⁻, CH₃CO₂ ⁻, or a dianion, namely, SO₄ ²⁻ orHPO₄ ²⁻, or a trianion namely PO₄ ³⁻ or from the group comprising C⁻,Br⁻, I⁻, acetate, lactate, citrate, tartrate, glycolate, glycerate,glutamate, β-hydroxyglutamate, glucouronate, gluconate, malate,aspartate, and more preferably from the group comprising Cl⁻, Br⁻, I⁻.

In a preferred sub-group of compounds of Formula I A and B are both—NR¹R² groups and these may be the same or different and R¹ and R² areselected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl,i-pentyl, n-hexyl, HO(CH₂)₂—, 2-ethylpiperidino, 2-methylpyrrolidino andcyclohexyl. In this preferred sub-group it is further preferred thatwhen R¹ and R² are both HO(CH₂)₂— in the A group that R¹ and R² in the Bgroup are both n-Pent.

In a further preferred sub-group of compounds of Formula I A and B maybe the same or different and R¹ and R² are selected independently fromethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl,2-ethylpiperidino, 2-methylpyrrolidino and cyclohexyl.

In a further preferred sub-group of compounds of Formula I A and B maybe the same or different and R¹ and R² are selected independently fromethyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino,2-methylpyrrolidino and cyclohexyl.

In a further preferred sub-group of compounds of Formula I A and B maybe the same or different and the sum of the carbon atoms in the alkylside chains represented by R¹ and R² is from 14 to 40, preferably from16 to 36, and more preferably from 18 to 30, and especially from 18 to24.

In a further preferred sub group of compounds of Formula I A and B maybe the same or different and the sum of the carbon atoms in the alkylside chains represented by R¹ and R² is from 16 to 20 preferably from 18to 20,

In one embodiment preferably A and B are the same and both R¹ and R² aren-propyl, n-butyl or n-pentyl.

In a further embodiment A and B are preferably different and R¹ and R²are the same or different, preferably selected from n-propyl, n-butyl,n-pentyl, n-hexyl, n-octyl, 2-methylpyrrolidino, 2-ethylpiperidino, andmorpholino.

Especially preferred moieties for use in treatments that requireremoval, deactivation or killing of unwanted tissues or cells are asfollows:

-   3,7-(tetra-n-propylamino)-phenothiazin-5-ium;-   3,7-(tetra-n-butylamino)-phenothiazin-5-ium;-   3,7-(tetra-n-pentylamino)-phenothiazin-5-ium;-   3,7-(tetra-iso-pentylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3,7-(N,N-tetra-iso-butylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;-   3-(N,N-diethanol amino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3-(N,N-diethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-butylamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;    and-   3-((N-ethyl-N-cyclohexyl)amino)-7((-N-ethyl)-N-cyclohexyl)amino-phenothiazin-5-ium.    These compounds preferably include a halide as a counteranion which    is preferably C⁻, Br⁻ or I⁻.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Symmetrical phenothiazinium salts: in vivo activity at 1 h.Figure shows % cross sectional tumour necrosis at 72 h post-PDT. Alldrugs were administered i.v. at a dose of 16.7 μmol/kg. At 1 h post drugadministration light (60 J/cm², 50 mW/cm²) was administeredsuperficially.

FIG. 2. Area of tumour necrosis (expressed as a % total section area) 72hrs after PDT with tetra-n-propyl and tetra-n-pentyl derivative (16.7μmolkg⁻¹, 660 nm light @50 mWcm⁻², 60 Jcm⁻²), Data points representmean+SEM (n=6, each reading measured in triplicate).

FIG. 3. Skin photosensitivity-murine ear swelling response. CBA/Gy micewere injected with sensitizer at 16.7 μmol/kg. At 24 h post druginjection ears were exposed to broad band white light from a xenon arclamp (25 J/cm², 30 mW/cm²). % Change in ear thickness was measured as:(ear thickness at 24 h post illumination−ear thicknesspre-illumination)/ear thickness pre-illumination×100. Increased % changein ear thickness measures increased skin photosensitivity.

FIG. 4. Log change in CFU/ml of E. coli incubated for 30 minutes with 10μM phenothiazine and illuminated for 60 minutes at 1.3 mW/cm⁻².

FIG. 5. Log change in CFU/m of E. coli incubated for 30 minutes withdifferent concentrations of tetra butyl phenothiazine and illuminatedfor 15 minutes at 1.3 mW/cm².

FIG. 6. Log change in CFU/ml of E. coli in the stationary phase ofgrowth following incubation for 30 minutes with 10 μM phenothiazine andilluminated for 60 minutes at 1.3 mWcm⁻².

FIG. 7. Log change in CFU/ml of E. coli resuspended in nutrient media.Cell were incubated for 30 minutes with 10 μM phenothiazine andilluminated for 60 minutes at 1.3 mW/cm⁻².

FIG. 8. Log change in CFU/ml of E. coli following incubation with 10 μMphenothiazine for 30 minutes. Illuminated was with laser light (664 nm)for 4 minutes at 0.1 W.

FIG. 9. Update of 10M phenothiazine into E. coli cells following a 30minute incubation. Cells were washed twice in 0.1M pH7.0 potassiumphosphate buffer to remove extra-cellular or loosely bound sensitiser.

FIG. 10. Log change in CFU/ml of E. coli cells incubated with 10 μMtetra butyl phenothiazine. Cells were washed twice with 0.1M pH7potassium phosphate buffer. Illuminated used laser light (664 nm) at 0.1W for 4 minutes.

FIG. 11. Percentage growth of a culture of an E. coli culture ascompared to a control when 10 μM phenothiazine was included in thegrowth media. Incubation was carried out in the dark at 37° C. for 6hours. Measurements based on apparent turbidity at 550 nm.

FIG. 12. Change in absorbance of an E. coli grown in the presence of 10μM tetra butyl phenothiazine in the light and in the dark.

FIG. 13. Percentage cell survival of P. aeruginosa following incubationwith 10 μM tetra butyl phenothiazine. Illumination was with laser light(664 nm) at 0.1 W.

FIG. 14. Percentage cell survival of S. aureus following incubation with10 μM tetra butyl phenothiazine. Illumination was with laser light (664nm) at 0.1 W.

FIG. 15. Percentage cell survival of MRS following incubation with 10 μMtetra butyl phenothiazine. Illumination was with laser light (664 nm) at0.1 W.

FIG. 16. Percentage cell survival of C. albicans following incubationwith 10 μM tetra butyl phenothiazine. Illumination was with laser light(664 nm) at 0.1 W.

FIG. 17. A graph depicting the percentage change in ear thickness at 24hours post-light.

Protocol: Drug applied topically, dose=5.79 g (20 μl at 0.5 mM)

Applied light dose=25 J/cm² (30 mW/cm² for 831 sec).

The phenothiazinium compounds used in the present inventions weresynthesised in Leeds University Department of Colour Chemistry under thedirection of J. Griffiths.

In outline the phenothiazines of Formula I may be synthesised asfollows:

1) Symmetrical phenothiazinium compounds where A=B

a) Phenothiazine is firstly brominated with bromine in glacial aceticacid to give 3,7-dibromophenothiazin-5-ium bromide, the suspensionformed is collected by filtration.

b) the 3,7-dibromophenothiazin-5-ium bromide is added to an aminerepresented by R′R″NH (in which R′ and R″ are as defined above) orN-heterocycle in chloroform. The solid formed is collected by filtrationand purified for example by flash column chromatography over silica gel60, using chloroform, chloroform/methanol (98/2) and thenchloroform/methanol (90/10). The product may be further purified byprecipitation from chloroform with petroleum ether (b.p. 60-80° C.).2) Unsymmetrical phenothiazinium compounds where A Ba) Phenothiazine in chloroform is cooled to below 5° C. and a solutionof iodine in chloroform added. The solid formed is collected byfiltration, washed with chloroform until free of iodine and then kept atroom temperature under vacuum overnight to give phenothiazin-5-iumtetraiodide hydrate.b) the phenothiazin-5-ium tetraiodide hydrate in methanol is added to asolution an amine R′R″NH in methanol (in which R′ and R″ are as definedabove). The reaction mixture is stirred overnight, reduced byevaporation left to cool. The solid that formed is collected byfiltration, washed with diethyl ether and dried.c) triethylamine in dichloromethane followed by a solution of adifferent second amine R′R″NH (in which R′ and R″ are as defined above)in dichloromethane is added to a solution of the solid from b) above indichloromethane. The reaction mixture is stirred overnight, the organiclayer washed with dilute hydrochloric acid and water, separated anddried (MgSO₄). The majority of the solvent is evaporation and diethylether added to precipitate the product which is collected by filtration,washed with diethyl ether and dried. Further purification of theproduct, if necessary, may be by flash column chromatography.

The present invention also provides compositions comprising a compoundof Formula I together with a diluent or excipient. Further compositionsof the present invention include those comprising two or more compoundsof Formula I and those comprising one or more compounds of Formula Iwith one or more different therapeutic or active agents.

The compounds of Formula I may be formulated into a variety ofpharmaceutical compositions which contain the compounds andpharmaceutically acceptable carriers, excipients, adjuvants (eachselected for certain characteristics that permit optimal formulation ofa pharmaceutical composition). The compositions include liposomes,nanoparticles, colloidal suspensions, micelles, microemuisions, vesiclesand nanospheres.

The compositions may also comprise further components such asconventional delivery vehicles and excipients including solvents such asalcohols (for example ethanol, polyethylene glycol, glycerol orn-butanol), dimethyl sulphoxide, water, saline, solubilisers such ascastor oil derivatives for example ethoxylated castor oils likeCremophor EL (trade mark BASF AG) or Tween (trade mark, ICI AmericasInc.) types, isotonising agents such as urea, glycerol, aminoethanol,propylene glycol, pH regulators, dyes, gelling agents, thickeners,buffers, and combinations thereof.

Typically the compositions are prepared by mixing a compound of FormulaI with one or more pharmaceutically acceptable carriers at anappropriate temperature, typically from 15° to 65° C. at an appropriatepH, typically from pH 3 to 9 and preferably at a physiologicallyacceptable pH such as from pH 6.5 to 7.5.

The concentration of the compounds of the present invention in thecompositions depends on the compound's photosensitising ability and ispreferably in the range from 0.0005 to 20% for topical use and from 100μM to 30 mM for intravenous use.

Dry compositions, which may be reconstituted before use, are alsoprovided in the present invention. These may be prepared by dry mixingsolid components of the composition or preparing a liquid compositionwhich is evaporated to dryness generally under mild conditions undervacuum or in low temperature ovens.

According to a further feature of the present invention the compounds ofFormula I have many uses, particularly in the treatment of infection andcancer; the treatment of dermatological, ophthalmic, cardiovascular,gynecological and dental conditions; and the prevention of infection,The uses may be in humans or animals. The present compounds may be usedagainst microorganisms.

In one embodiment of the present invention a compound of Formula I isused as a PDT agent or a photodiagnostic agent.

In one embodiment of the present invention compounds of Formula I andcompositions comprising them are used as medicaments particularly asanticancer agents, as antibacterials, antifungals and antivirals.

Examples of uses of the compounds of the present invention are asphotosensitising drugs for PDT to treat cancer and pre-cancerousconditions including Barrett's oesophagus and cervical intraepithelialneoplasia (CIN), bladder cancer, colon cancer, non-melanoma skin cancer,actinic keratoses, melanoma, brain-pituitary cancer, brain-glioma,pancreatic cancer, head and neck cancer, lung cancer, particularly nonsmall cell, mesothelioma, oesophageal cancer, stomach cancer, cutaneousT-cell lymphoma; to treat infections, for example for use asanti-microbial and antifungal treatments for skin and wound infectionssuch as burn wounds, in treatment of ulcers particularly leg ulcers moreparticularly infected chronic leg ulcers, nail infections; for parasiticinfection, stomach infection, malaria, leprosy, for bacterial and fungalspore inactivation, for treatment of prions and viral infection such asHIV, for ear, nose and throat infections, tuberculosis, sexuallytransmitted diseases (STD's), herpes, for treatment of Candida localisedinfections for example of hair, nails and epidermis, such as tinea pedisand candida vulvovaginitis; and for use as infection preventatives suchas sterilisation of surgical wounds, skin graft sterilisation, stem cellsterilisation, graft versus host disease; to treat opthalmologicalconditions such as macular degeneration, occult choroidalneovascularisation (CNV), CNV due to pathological myopia, occult withage related macular degeneration (AMD), diabetic macular oedema,vascular problems such as cardiovascular disease, arteriosclerosis andrestenosis and autoimmune diseases such as rheumatoid arthritis, skindiseases such as psoriasis, acne, vitiligo and eczema and otherdermatological conditions such as hirsuitism, and sun damage, otherbenign conditions such as endometriosis and menorrhagia.

The compounds may also be used for other local infections as well as inthe treatment of dental bacterial disease, such as gum abscesses, gumdisease, gingivitis, and removal, deactivation or killing of plaquebiofilms. The compounds may also be used in photochemicalinternalisation (the use of photosensitisers to assist the uptake andsubcellular localisation of drugs) through their photosensitisingproperties and in non-therapeutic uses such as in photodiagnosis throughtheir fluorescence properties. The latter approach takes advantage ofthe fact that the photosensitiser concentrates more in tumours than insurrounding healthy tissue and when induced to fluoresce (by applicationof blue light), the tumour fluoresces more strongly than the surroundingtissue. Examples of applications areas include diagnoses for oraldiseases and for diseases of the bladder, lung and skin.

In addition to the above the present compounds are used asphotosensitising drugs for PDT in veterinary applications, for examplein treatment of cancers such as ear cancer in cats, as antifungal,antibacterial and antiviral treatments, for sterilisation of wounds inanimals and for opthalmological treatments in animals.

The use of the compounds of Formula I is preferably in treatments oflocalised and/or early cancer and/or pre-cancerous lesions in humans andin animals; or in the treatment and/or prevention of infections inwounds or skin in humans and animals.

According to a further feature of the present invention the presentcompounds may be used as photoactivated antimicrobial, antifungal andantiviral agents for sterilisation of surfaces and fluids, for examplethey may be used to sterilise surgical implants and stents, particularlywhere these are coated or impregnated, to sterilise textiles such asbandages and dressings, IV lines and catheters, for sterilisation ofwater, air, blood, blood products, and food and food packaging toprevent transfer of infection, and for general household, hospital andoffice cleaning. The compounds are preferably used to sterilise surgicalimplants and stents, particularly where these are coated or impregnated,to sterilise textiles such as bandages and dressings, IV lines andcatheters, for sterilisation of water, air, and food and food packagingto prevent transfer of infection, and for general household, hospitaland office cleaning. The compounds may be applied to or contacted withthe surfaces and fluids and activating the compound by exposure tolight. Additionally the surface to be sterilised may be immersed in amixture or solution of the compound or the fluid to be sterilised may bemixed with the compound or a solution or mixture containing thecompound.

The present invention relates to phenothiazinium sensitizers which showan unexpected and pronounced dependence of their photobiologicalproperties in vitro and in vivo on the size and hydrophobic character ofsubstituents on the terminal amino groups. By careful selection of suchstructural features, photosensitisers with distinct advantages overexisting materials are provided. Accordingly compounds of the presentinvention overcome the problems of the prior art by providing thefollowing advantages in the field of oncology and in their antimicrobialeffects:

Advantages in Oncology

-   -   Extremely strong photoactivity when compared with methylene and        ethylene blue.    -   Low absorption of light in the UV/blue region. This results in a        lower propensity of the compounds to skin photosensitivity.    -   Rapid skin clearance.    -   High selectivity for tumours.    -   Low dark toxicity.    -   Low potential for DNA damage when compared with methylene blue.    -   Very short drug-to-light time interval compared with existing        PDT drugs.        Antimicrobial Advantages    -   Highly effective in deactivating a wide range of microorganisms,        including Gram positive and Gram negative bacteria, MRSA and        fungal infection.    -   Active against quiescent/stationary bacteria.    -   High selectivity for microorganisms with minimum damage to host        tissue.    -   Unexpectedly low level of photobleaching.

In any of the uses described above the compounds of the presentinvention may be used advantageously in mixtures comprising two or morecompounds of Formula I and in mixtures comprising one or more compoundsof Formula I with one or more different therapeutic or active agents.

The compounds of the present invention are particularly useful asphotosensitising drugs for PDT of conditions where treatment requiresremoval, deactivation or killing of unwanted tissue or cells such ascancer, precancerous disease, ophthalmic disease, vascular disease,autoimmune disease, and proliferative conditions of the skin and otherorgans. Specific and unpredicted advantages of these materials relate totheir ability to be photoactive against target tissues at differenttimes after systemic administration (depending upon the particularsensitiser used) and therefore their ability to be targeted directly forexample to the vasculature or tumour cells. They also have a lowtendency to sensitise skin to ambient light when administeredsystemically and a low tendency to colour skin.

Accordingly, the present invention provides a method of treatment forcancer and other human or animal diseases through systemic or localadministration of the photosensitiser, followed by application of lightof an appropriate dose and wavelength or wavelength range.

For the present compounds activation is by light, including white light,of an appropriate wavelength, usually in the range from 600 to 800 nm,preferred wavelengths are from 630 nm to 700 nm.

The light source may be any appropriate light source such as a laser,laser diode or non-coherent light source.

The light dose administered during PDT can vary but preferably is from 1to 200 J/cm², more preferably from 20 to 100 J/cm².

Light exposure may be given at any time after a drug is initiallyadministered or up to 48 hours after drug administration and the timemay be tailored according to the condition being treated, the method ofdrug delivery and the specific compound of Formula (I) used. Lightexposure is preferably given at any time after a drug is initiallyadministered up to 3 hours, more preferably from the time after a drugis initially administered up to 1 hour, especially up to 10 minutes.

Increased intensity of the light dose generally reduces exposure times.

It is preferred that exposure to light is localised to the area/regionto be treated, and where tumours are being treated more preferablylocalised to the tumour itself.

In one embodiment of the present invention the compound is preferablyadministered to a subject in need of treatment is that according toformula (I), where R¹ and R² are n-propyl and said light exposure isgiven up to 10 minutes after a drug is initially administered.

In a further preferred embodiment of the invention, light exposure isgiven within 1 minute after a drug is initially administered.

More preferably light exposure is given at the point of drugadministration.

Where the compound administered is that according to Formula (I), whereR¹ and R² are n-pentyl said light exposure is given at longer times, forexample up to 60 minutes after a drug is initially administered.

Where the compounds of the present invention are used as PDT agents formammalian cells and tumours they may be administered using the abovedescribed compositions in a variety of ways, such as systemically orlocally and may be used alone or as components or mixtures with othercomponents and drugs. Where administered systemically the compounds maybe delivered for example intravenously, orally, sub-cutaneously,intramuscularly, directly into affected tissues and organs,intraperitoneally, directly into tumours, intradermally or via animplant.

Where administered locally or topically the compounds may be deliveredvia a variety of means for example via a spray, lotion, suspension,emulsion, gel, ointment, salves, sticks, soaps, liquid aerosols, powderaerosols, drops or paste.

The compounds of the present invention have the advantage, compared withother phenothiazinium photosensitisers, that they do not, in carryingout their cell-destroying activity, target the nucleus of the cell sothat there is a much lower risk of the cells undergoing mutagenictransformations.

The dose rates of the compounds of Formula I for intravenousadministration to humans for oncology treatments are in the range 0.01to 10 μmol (micromole)/kg, preferably in the range 0.1 to 2.0 μmol(micromole)/kg. To achieve a dose of say 2 μmol (micromole)/kg in a 70kg patient requires injection of 70 ml of a 2 mM solution, or 5 ml at aconcentration of 27 mM (16 mg/ml) or 2.8 ml of a 50 mM solution. Typicalinjections volumes are in the range 0.1 to 100 ml, preferably from 5 to50 ml.

In one embodiment the method for treatment of cancer comprises the stepof administering a compound according to Formula (I) where R¹ and R² areselected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl,i-pentyl, n-hexyl, 2-ethylpiperidino, 2-methylpyrrolidino and HO(CH₂)₂,preferably where R¹ and R² are selected independently from ethyl,n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl,2-ethylpiperidino and 2-methylpyrrolidino.

In a further feature of the present invention the compounds of Formula Ipreferably are used against bacteria, more preferably the compounds areused against antibiotic resistant bacteria.

The compounds may also be used in PDT as photoactivatableantimicrobials, including antibacterials, antifungals or antivirals,treatments for skin and other local infections, for sterilisation ofburn wounds and other lesions, for sterilisation of both recipienttissue and donated tissue during organ, including skin, transplantationand for the treatment of dental microbial disease.

The said compounds are also useful as photoactivatable antimicrobialagents for general sterilisation of surfaces and fluids. Specificadvantages of these compounds over existing known antimicrobialphotosensitisers are their high photocytotoxicity at low light levels,combined with a low tendency to undergo photobleaching.

According to a further feature of the present invention there isprovided a method of treatment of microbial infections, burn wounds andother lesions and of dental bacterial disease, the method comprisingsystemic administration or applying to the area to be treated (forexample by a spray, lotion, suspension, emulsion, ointment, gel orpaste) a therapeutically effective amount of a compound of the presentinvention and exposing said area to light to render active saidcompound.

In one embodiment the method comprises the step of administering acompound according to Formula I in which A and B are both —NR¹R² groupsand these may be the same or different where R¹ and R² are selectedindependently from ethyl, n-propyl, n-butyl, n-pentyl, i-pentyl,n-hexyl, 2-ethylpiperidino, 2-methylpyrrolidino and cyclohexyl.

In a further embodiment the method comprises the step of administering acompound according to Formula (I) in which A and B are both —NR¹R²groups where R¹ and R² are n-butyl or n-pentyl.

According to a further feature of the present invention there isprovided a method of prevention of microbial infections, for example inwounds, surgical incisions, burn wounds, and other lesions and of dentalbacterial disease, the method comprising systemic administration orapplying to the area to be treated (for example by a spray, lotion,suspension, emulsion, ointment, gel or paste) a therapeuticallyeffective amount of a compound of the present invention and exposingsaid area to light to render active said compound. The compounds ofFormula I may be applied to prevent infection at any stage includingwound contamination, where non-replicating organisms are present in awound; wound colonisation where replicating microorganisms are presentin a wound; and wound infection where replicating microorganisms arepresent that cause injury to the host. When there are >10⁵ CFU/g tissue,it is more likely that sepsis will develop.

The concentration used for bacterial cell kill in vitro is in the rangefrom 0.1 to 100 μM, preferably from 1 to 50 μM and more preferably from5 to 20 μM, especially 10 μM.

Furthermore, the present invention provides a conjugate or compositeformed between a compound of Formula I and a polymer. The term compositeas used herein refers to the situation wherein a compound of theinvention is embedded in a polymer or physically occluded within oradsorbed onto a matrix or substrate. The polymer may be a biologicalpolymer such as a peptide or a protein. Preferred polymers include thosehaving anhydride and/or ester groups. Preferred compounds of Formula Iwhich form a conjugate or composite with a polymer are those in which atleast one of the Group A or B is a piperazinyl group.

In addition, the present invention provides a compound formed by thereaction between a compound of Formula I and a chlorotriazinederivative. The chlorotriazine derivative may be a polymer havingchlorotriazine groups attached thereto.

Appropriate compounds of the present invention may be attached topolymeric surfaces, permanently by covalent bonds or reversibly byintermolecular interactions, thus affording a surface that can besterilised whenever required by the application of light. This would beuseful, for example, with intravenous lines in patients and in suturesand catheters and intravenous lines, where maintaining long-termsterility of the relevant surfaces is problematical. The resistance ofthe compounds to photobleaching is an advantage in such applications,where prolonged stability of the chromophore is required.

Accordingly the present invention also provides an article having atleast one surface to which is attached a compound of the presentinvention.

Preferably the article is a medical device such as a venous, urinary orballoon catheter, suture, orthopedic or artificial implant, heart valve,surgical screw or pin, pacemaker lead, feeding or breathing tube,vascular stent, intraocular lens, or small joint replacement. Thearticle may also be of use in wound care and for packaging materials formedical use, for example, materials for medical equipment.

A compound of the present invention may be applied to or contacted withwalls, floors and ceilings of hospitals, clinical surfaces such asoperating tables, abattoirs, clean rooms in scientific laboratories,fibres which may be converted into woven, knitted or non-woven textilearticles such as cleaning cloths, wipes, surgical gowns, bed linen,wound dressings and bandages. The compound may be applied directly orvia attachment to a polymeric species.

Where the compound is to be applied to walls, floors, ceilings, and worksurfaces, it is envisaged that it will be used as a component of a paintor lacquer, which comprises the compound, film forming polymers, whichmay or may not be cross-linkable, and an appropriate solvent, optionallywith drying agents and other colorants. The surface coating may take theform of a solution or water-based dispersion.

Where compound or polymer is applied to walls, floors, ceilings this maybe via a surface coating such as a paint:

Alternatively the article is one for use in the food and beverageindustry and may be an item of packaging, a wrapper or storage carton ora piece of processing equipment.

The article may be a refrigerator, vending machine, ice making machine,a piece of restaurant equipment or other kitchen appliance.

Furthermore, the present invention also provides a method of sterilisinga surface or a fluid comprising contacting or applying the compound ofthe present invention to said surface or fluid and activating saidcompound by means of light. The compound may be contacted or applied byany means, for example as a spray, liquid, solution, suspension, foam,cream, gel or emulsion.

According to a further feature of the present invention there isprovided a method for sterilising fluids in which the fluid is contactedwith a compound of Formula I or with a conjugate or composite formedbetween a compound of Formula I and a polymer whilst the compound or theconjugate or composite is illuminated.

The fluid may be a liquid or a gas or a vapour. The method may forexample be applied to sterilisation of liquids, for example forsterilisation of water, or liquids used medically such as parenteralliquids for example saline or glucose and particularly for sterilisationof biological liquids such as blood, blood products, red cells, bonemarrow cells, and stem cells. The method may also be applied tosterilisation of gases such as air, particularly air used in airconditioning systems, and oxygen used medically. This method isparticularly useful for sterilising materials which cannot be easilysterilised by filtration methods.

The method is used preferably for sterilisation of water, or liquidsused medically such as parenteral liquids such as saline or glucose andfor sterilisation of biological liquids such as bone marrow cells andstern cells.

The compounds of Formula I and its conjugates or composites may be usedas is, preferably with its surface area maximised such as in a finelydivided form or in the form of beads or plates, or it may be used on orassociated with any support material which provides a large surface areasuch as glass, glass wool, ceramics, plastics, metals and metal oxides.The support material is preferably transparent to light or allows lightto pass through it. Where a support material is used this is arranged tomaximise the surface area covered by the conjugate or composite and maybe in the form of beads, plates, large surface areas in columns ortubes, foams or fibres.

The compound of Formula I or its conjugate or composite is preferablycontinuously illuminated at the wavelengths and at the light dosesdescribed above.

The preferred compounds of Formula I are those preferred in thissterilisation method.

In a particular embodiment of this aspect of the invention the compoundof Formula I or its conjugate or composite either alone or on a supportmaterial is packed into a column, typically made of a material which istransparent to light, such as silica glass or synthetic fibres. Thefluid requiring sterilisation is passed into one end of the column, thewhole column is continuously illuminated and sterilised material flowsout from the other end of the column.

Certain compounds of Formula I wherein:

A and B each independently is

in which R′ and R″ each independently is a linear, branched or cyclichydrocarbon group, or R′ and R″ together with the N atom to which theyare attached form an optionally substituted 5-, 6- or 7-membered ring;and where X^(P−) is a counteranion and P is 1, 2 or 3;except for the compounds in which A and B are the same and are selectedfrom —N(CH₃)₂, —N(CH₂CH₃)₂, N(n-Pr)₂, —N(n-Bu)₂, —N(n-Pent)₂,—N(n-Hex)₂, —N(n-Hept)₂, piperidino, —N(CH₂CH₂OH)₂, —N(diethylhexyl)₂,and not including those in which A is selected from —N(Me)₂ or —N(Et)₂and B is selected from —N(CH₂CH₂OH)₂, piperidino, morpholino,thiomorpholino, —N(Et)₂, —N(MeEt), —N(Me)₂ are novel and accordinglythese form a further feature of the present invention. The preferencesdescribed above apply to the compounds of Formula I themselves.

Specific novel moieties include:

-   3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3,7-(N,N-tetra-iso-butylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;-   3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-   3-(N,N-diethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-   3-(N,N-di-n-butylamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;    and-   3-((N-ethyl-N-cyclohexyl)amino)-7((-N-ethyl)-N-cyclohexyl)amino-phenothiazin-5-ium    These compounds preferably include a halide as a counteranion which    is preferably Cl⁻, Br⁻ or I⁻.

EXAMPLES

1) General synthesis of symmetrical phenothiazinium bromides of FormulaI where (A=B=R′R″N— or A=B=N-heterocycle; p=1, X^(p−)=Br⁻).

a) Preparation of 3,7-dibromophenothiazin-5-ium bromide

To a solution of phenothiazine (2.00 g, 0.01 mol) (Note 1) inoxygen-free, glacial acetic acid (150 cm³) was added, in one portion andwith vigorous stirring, a solution of bromine in oxygen-free, glacialacetic acid (100 cm³, 10% v/v Br₂). The reaction mixture became darkwith the formation of a dark solid. Stirring was continued for oneminute and water (400 cm³) was then added, when the suspension took on ared appearance. The reaction mixture was vacuum filtered to produce adark solid and a brown filtrate. The solid was washed with ether anddried under vacuum (40° C., 50 mmHg) for one hour to yield a brick redproduct. Mass of solid=3.63 g Yield=83%.

b) Preparation of the symmetrical phenothiazinium bromides

To a solution of the appropriate amine R′R″NH or N-heterocycle (32.4mmol) in chloroform (200 cm³) under nitrogen and with vigorous stirringwas added, in one portion, 3,7-dibromophenothiazin-5-ium bromide (2.0 g,4.6 mmol). The reaction mixture became blue in colour and was stirredunder nitrogen for 3 hours. The chloroform solution was washedsuccessively with HBr (2% aq., 2×50 cm³) and water (2×50 cm³), and thendried over MgSO₄. After filtration, the majority of the solvent wasremoved by rotary evaporation, an excess of diethyl ether was added andthe reaction mixture then left to stand. After some time, a large amountof colourless solid was deposited. This material was removed byfiltration. The filtrate was evaporated to dryness and the residualcrude product was purified by flash column chromatography over silicagel 60, employing sequentially a mobile phase of chloroform,chloroform/methanol (98/2) and finally chloroform/methanol (90/10). Therelevant blue chromatographic fractions were combined and the solventremoved by rotary evaporation. The dark blue product was taken up in aminimum volume of dichloromethane (10 cm³) and the final productprecipitated in crystalline form by the addition of an excess ofpetroleum ether (b.p. 60-80° C.). The solid was collected by filtration,washed with ether and air dried.

The purity of each product was confirmed by thin layer chromatography(showing a single detectable blue spot), and the structure was confirmedby electrospray mass spectrometry and UV/visible absorptionspectroscopy).

2) General synthesis of unsymmetrical phenothiazinium iodides of FormulaI where (A≠B=R′R″N or N-heterocycle, p=1, X^(p−)=I⁻).

a) Preparation of phenothiazin-5-ium tetraiodide hydrate

To a stirred solution of phenothiazine (10 mmole) in chloroform (100cm³) cooled to below 5° C. in an ice bath was added, over 1.5 hours, asolution of iodine (33 mmole) in chloroform (400 cm³). The mixture wasstirred for 30 minutes and the resultant precipitate was collected byfiltration, washed with chloroform until free of iodine and then kept atroom temperature under vacuum overnight to give the product.

b) Preparation of the unsymmetrical phenothiazin-5-ium iodides

To a stirred solution of phenothiazin-5-ium tetraiodide hydrate (1.4mmole) in methanol (300 cm³) was added, dropwise, over a period of 60minutes a solution of the appropriate amine R¹R²NH (3.6 mmole) inmethanol (50 cm³). The reaction mixture was stirred overnight. Thevolume of the reaction mixture was then reduced by evaporation and thehot solution left to cool. The solid that formed was collected byfiltration, washed with diethyl ether and dried.

c) To a solution of this solid (0.34 mmol) in dichloromethane (100 cm³)was added a solution of triethylamine (0.40 mmol) in dichloromethane (5cm³) followed by a solution of a different second amine R′R″NH (1.4mmol) in dichloromethane (50 cm³) over 60 minutes. The reaction mixturewas stirred overnight. The organic layer was then washed with dilutehydrochloric acid (4×25 cm³) followed by water (2×25 cm³). The organiclayer was then dried (MgSO₄). The majority of the solvent was removed byrotary evaporation and an excess of diethyl ether added to precipitatethe solid. The solid was collected by filtration, washed with diethylether and dried.

Further purification of the compound, if necessary, was by flash columnchromatography.

The purity of each product was confirmed by thin layer chromatography (asingle detectable blue spot). Structures were confirmed by electrospraymass spectrometry and UV/visible absorption spectroscopy.

The following specific compounds were prepared by the above methods:

Compound 1 R¹-R⁴=n-C₃H₇: tetra-n-propylCompound 2 R¹-R⁴=n-C₄H₉: tetra-n-butylCompound 3 R¹-R⁴=n-C₅H₁₁: tetra-n-pentylCompound 4 R¹-R⁴=n-C₆H₃: tetra-n-hexyl

Methylene blue (R¹-R⁴=n-CH₃) Compound 5 and ethylene blue (R¹R⁴=n-C₂H₅)Compound 6 were also examined for comparative purposes. Compounds 1 to 6have iodide counteranions.

Compounds 7, 7a, 8, 8a, 8b and 14-31 were made by analogous methods.

Compound 93-(N,N-dimethylamino)-7-(N,N-dipropylamino)-phenothiazin-5-ium iodide(20%)

This compound was obtained following isolation of3-(N,N-dipropylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with dimethylamine hydrochloride. Precipitation fromdichloromethane by addition of diethyl ether yielded purple lustrouscrystals. Mass spectrometry: C₂₀H₂₆N₃OS requires m/z=340; found m/z=340(I⁻ not detected by mass spectrometry).

Compound10—3-(N,N-diethylamino)-7-(N,N-dipropylamino)-phenothiazin-5-ium iodide(15%)

This compound was obtained following isolation of3-(N,N-dipropylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with diethylamine. Precipitation from dichloromethane byaddition of diethyl ether yielded purple lustrous crystals. Massspectrometry: C₂₂H₃₀N₃OS requires m/z=368; found m/z=368 (I⁻ notdetected by mass spectrometry).

Compound 113-(N,N-dibutylamino)-7-(N,N-dipropylamino)-phenothiazin-5-ium iodide(19%)

This compound was obtained following isolation of3-(N,N-dipropylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with dibutylamine. Precipitation from dichloromethane byaddition of diethyl ether yielded purple lustrous crystals. Massspectrometry: C₂₆H₃₅N₃OS requires m/z=424; found m/z=424 (I⁻ notdetected by mass spectrometry).

Compound 123-(N,N-dipentylamino)-7-(N,N-dipropylamino)-phenothiazin-5-ium iodide(20%)

This compound was obtained following isolation of3-(N,N-dipropylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with dipentylamine. Precipitation from dichloromethane byaddition of diethyl ether yielded purple lustrous crystals. Massspectrometry: C₂₈H₄₂N₃OS requires m/z=452; found m/z 452 (I⁻ notdetected by mass spectrometry).

Compound 133-(N,N-dihexylamino)-7-(N,N-dipropylamino)-phenothiazin-5-ium iodide(22%)

This compound was obtained following isolation of3-(N,N-dipropylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with dihexylamine. Precipitation from dichloromethane byaddition of diethyl ether yielded purple lustrous crystals. Massspectrometry: C₃₀H₄₆N₃OS requires m/z=480; found m/z=480 (I⁻ notdetected by mass spectrometry).

Compound 283-(N,N-diethanolamino)-7-(N,N-dipentylamino)-phenothiazin-5-ium iodide(23%)

This compound was obtained following isolation of3-(N,N-dipentylamino)-phenothiazin-5-ium triiodide and subsequenttreatment with diethanolamine. Precipitation from dichloromethane byaddition of diethyl ether yielded purple lustrous crystals. Massspectrometry: C₂₆H₃₈N₃O₂S requires m/z=456; found m/z=456 (I⁻ notdetected by mass spectrometry).

Further compounds have been synthesised and these are summarised inTable A.

Compounds 5 and 6 are not compounds of the present invention and areincluded for comparative purposes.

Stock solutions of photosensitisers were made up in water and/or DMSOand stored in the dark until required. Test solutions were made up inbuffer or solvent or biological medium as required.

Spectral and Physical Properties of the Phenothiazinium Compounds

Spectral data of the phenothiazinium compounds in water and in methanol(Table 1), show that all of the compounds have absorption peaks in the650-700 nm region, but that there is considerable variability in theprecise peak position. The phenothiazinium compounds with longer alkylchains have absorption peaks at longer wavelengths and the peakpositions in general are at longer wavelengths in water compared withmethanol. These differences probably reflect the aggregation state ofthe photosensitisers.

The octanol-water partition coefficients (p) for the variousphotosensitisers are shown in Tables 3 and 4, wherep=log(mg/ml in octanol)/(mg/ml in buffer).

The octanol buffer partition coefficient (p) determines thelipophilicity of the drag. As might be expected, the lipophilicityincreases with increasing value of R, but it should be noted that evenfor higher values of R, the compounds remain soluble in biologicalmedia.

TABLE 1 λmax in methanol R¹ R² R³ R⁴ (nm) 1 n-Pr n-Pr n-Pr n-Pr 656 2n-Bu n-Bu n-Bu n-Bu 661 3 n-Pent n-Pent n-Pent n-Pent 665 4 n-Hex n-Hexn-Hex n-Hex 668 5 n-Me n-Me n-Me n-Me 669 6 n-Et n-Et n-Et n-Et 669 7i-Bu i-Bu i-Bu i-Bu 668 8 i-Pent i-Pent i-Pent i-Pent 662 9 Me Me n-Prn-Pr 659 10 Et Et n-Pr n-Pr 661 11 n-Bu n-Bu n-Pr n-Pr 665 12 n-Pentn-Pent n-Pr n-Pr 665 13 n-Hex n-Hex n-Pr n-Pr 666 14 n-Bu n-Bu n-Pentn-Pent 660 15 n-Bu n-Bu i-Pent i-Pent 661 16 Et Et n-Hept n-Hept 661 17Me n-Oct Me n-Oct 655 18 Et Cyclohex Et Cyclohex 668 19 piperidinopiperidino 667 20 2-ethylpiperidino n-Pent n-Pent 668 21 2-methylpyrrolidino n-Pent n-Pent 663 22 Morpholino Morpholino 660 23 Morpholinon-Pr n-Pr 663 24 Morpholino n-Bu n-Bu 661 25 Morpholino n-Pent n-Pent663 26 HO(CH₂)₂ HO(CH₂)₂ n-Pr n-Pr 663 27 HO(CH₂)₂ HO(CH₂)₂ n-Bu n-Bu660 28 HO(CH₂)₂ HO(CH₂)₂ n-Pent n-Pent 663 29 PhCH₂ PhCH₂ PhCH₂ PhCH₂649 Ex. Max is the fluorescence excitation wavelength maximum and Em.Max is the fluorescence emission wavelength maximum

Me, Et, Pr, Bu, Pent, Hex, Hept, Oct in the above table and throughoutthis specification represent methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl and octyl respectively, and that n- and i-indicate normaland iso alkyl chains.

The phenothiazinium derivatives were assessed for PDT efficacy in aseries of mammalian cells in culture. RIF-1 murine fibrosarcoma cellswere studied, using the MTT assay to assess remaining cell viabilityfollowing PDT. The data from a series of experiments are summarised inTable 2 in which R in the Table represents R¹ and R², which shows theLD₅₀ values (concentration of photosensitiser needed to kill half of thecells under the conditions used) for four of the phenothiazinederivatives. Also shown for comparison are the data for methylene blueand ethylene blue. Some of these compounds are also able to kill cellsin the dark and Table 2 also shows the ratio to LD₅₀ for dark onlycontrols. It may be seen from Table 2 that the tetra-n-pentylderivative, the tetra-n-butyl derivative and the tetra-n-propylderivative are all efficient PDT agents under these conditions, beingmuch more active than methylene blue or ethylene blue. The mostefficient is the tetra-n-propyl derivative. Also, it is clear that,while for methylene blue there is only a small ratio between the LD₅₀for dark and light toxicity, this ratio is much greater for thephenothiazinium derivatives. The results illustrate the much increasedphotoactivity of these compounds, but also their relative lack of darktoxicity compared with methylene blue. This is a considerable advantagein therapeutic terms. Table 2 also shows the relative activity of thevarious derivatives in comparison with methylene blue and ethylene blueusing a measure of their intrinsic ability to produce singlet oxygen. Itmay be seen that there is very little difference between the differentcompounds, showing that the marked differences observed in cells isalmost entirely due to biological factors, the mechanisms of which arenot yet known. Table 2 also shows the initial subcellular localisationof the different phenothiazinium compounds derivatives compared withthat of methylene blue and ethylene blue, as well as any re-localisationwhich occurs following light administration. It is noteworthy that,whilst all of the derivatives initially localise in lysosomes, whilstmethyene blue then relocalises to the nucleus (with possible deleteriousor mutagenic effects on DNA), the tetra-n-propyl, tetra-n-butyl,tetra-n-pentyl and tetra-n-hexyl derivatives relocalise to themitochondria, which is a much better PDT target.

Table 3 shows LD₅₀ values for some of the derivatives in a series ofdifferent cells in culture, representing different human tissues andcancers. It is clear that the tetra-n-propyl, the tetra-n-butyl and thetetra-n-pentyl derivatives are again highly active compared withmethylene blue and that they are also active in all cell lines tested.

Several asymmetric phenothiazinium derivatives (where R¹=R²≠R³=R⁴) havebeen prepared and tested in cells in culture. Several of these have beentested and shown to have superior properties as photosensitisers tomethylene blue, both in terms of absolute activity and in terms of thelight to dark toxicity ratio. Sample data for these compounds are shownin Table 4.

TABLE 4 Chemical properties of the asymmetric phenothiazines incomparison to methylene blue and phototoxicity and dark toxicity in SiHahuman cervical squamous cell carcinoma cells. Singlet Ratio λ_(max) inoxygen dark: methanol gener- PDT LD₅₀ PDT (nm) ation¹ Log P (μM)² LD₅₀ ²Methylene blue 656 47 −1.0 18.7 ± 1.0  1 (compound 5) Di-n-butyl 661 29+1.0 4.6 ± 2.0 7 morpholino (compound 24) Di-n-butyl 660 13 +1.3 1.7 ±0.3 87 diethanolamine (compound 27) Di-n-pentyl 663 32 +1.1 0.43 ± 0.0339 diethanolamine (compound 28) ¹% photo-oxidation of1,3-diphenylisobenzofuran after 10 min red light illumination with 100mg/ml of the phenothiazinium compounds in 90% DMF:10% water. ²Cells wereincubated with the phenothiazinium compounds for 2 h. For measurement ofphototoxicity, cells were illuminated with 3 J/cm² 665 nm light. Darktoxicity was measured in parallel. Cell survival was assessed after 48 husing the sulforhodamine B (SRB) assay.Anti-Tumour Efficacy In Vivo

Tumour destruction was assessed in CBA/gy mice bearing subcutaneous CaNTtumours. Photosensitiser was administered intravenously at doses up to16.7 μmol/kg. The dose was reduced to 8.35 μmol/kg if high levels ofmorbidity or mortality were observed or if solubility was limited. Atvarious times after photosensitiser administration, the tumour wasilluminated superficially with 60 J/cm2, 50 mW/cm2 light from a Patersonlamp using a 660±15 nm filter. Drug—light intervals ranged from 0 h (inpractice, 1-2 minutes) up to 96 h. 72 h after illumination a crosssectional slice was removed from the centre of the tumour parallel tothe incident light, an image of this was captured and the macroscopicnecrotic area quantified using image analysis software. Necrosis wasexpressed as % area of the total tumour slice. % tumour necrosis incontrol tumours was generally <10%. Antitumour activity was categorized:None 0-10% tumour necrosis, Low 11-39% tumour necrosis, Medium 40-69%tumour necrosis, High 70-100% tumour necrosis.

Antitumour activity at the optimal dose and drug—light interval for eachcompound is shown in Table 5.

TABLE 5 PDT induced tumour necrosis in CBA/gy mice following ivadministration at the optimal dose and drug - light interval, andPhotoinactivation of log phase E. coli and C. albicans using 10 μMphotosensitiser and illumination with 665 nm laser light, at a fluencerate of 3.2 J/cm², except for compounds 1, 4, 5, 6 which wereilluminated at a fluence rate of 1.3 J/cm². Oncology C. albicans CellDose Drug- Cell kill (Log₁₀ kill (Log₁₀ Counter Antitumour % Tumour(μmol/ light reduction in reduction in Compound anion activity necrosiskg) interval CFU/ml) CFU/ml) 5 Cl⁻ None  9 ± 4 16.7 1 h 0.55 (0.02) 6 I⁻None  2 ± 2 16.7 1 h 0.23 (0.02) 1 I⁻ High 93 ± 3 16.7 0 h 0.91 (0.12) 2Br⁻ High 95 ± 3 8.35 0 h 4.72 (0.30) 4.16 (0.14) 3 Br⁻ High 88 ± 6 1.671 h 5.29 (0.25) 3.61 (0.36) 4 Br⁻ Medium  47 ± 12 8.35 0 h I⁻ 0.09(0.02) 7 Br⁻ High 78 ± 3 16.7 0 h 2.56 (0.09) 4.15 (0.36) 8 Br High  74± 12 8.35 0 h 3.58 (0.13) 9 I⁻ Low 34 ± 2 16.7 0 h 2.82 (0.54) 10 I⁻Medium  45 ± 15 16.7 0 h 3.01 (0.14) 11 I⁻ High 98 ± 2 16.7 0 h 4.86(0.30) 12 I⁻ Low  38 ± 14 8.35 0 h 4.50 (0.28) 3.58 (0.97) 13 I⁻ High 85± 3 8.35 0 h 4.62 (0.34) 14 I⁻ Medium  60 ± 13 16.7 1 h 3.25 (0.58) 15I⁻ High 95 ± 3 16.7 0 h 2.84 (0.30) 5.81 (1.18) 16 I⁻ Low  25 ± 13 16.71 h 0.82 (0.01) 17 Br⁻ Medium  61 ± 11 16.7 0 h 0.23 (0.14) 18 Br⁻Medium  49 ± 14 16.7 0 h 4.74 (0.10) 2.41 (0.01) 19 I⁻ Low  36 ± 10 16.70 h 1.77 (0.03) 20 I⁻ High 86 ± 2 16.7 0 h 3.56 (0.27) 4.25 21 I⁻ High90 ± 4 8.35 0 h 3.21 (0.07) 22 I⁻ Low 17 ± 6 16.7 0 h — 23 I⁻ Low 14 ± 716.7 0 h 1.72 (0.30) 24 I⁻ None 10 ± 7 16.7 1 h 1.90 (0.36) 25 I⁻ Low 16± 5 16.7 1 h 1.70 (0.28) 26 I⁻ Low  17 ± 10 16.7 0 h — 27 I⁻ None  3 ± 216.7 0 h 0.8 28 I⁻ High 75 ± 4 8.35 0 h 1.5 29 I⁻ Medium  40 ± 17 16.7 1h 1.54 (0.02)

FIG. 1 shows the anti-tumour photodynamic efficacy (% tumour necrosis)of symmetrically substituted phenothiazines of type (I). Female CBA/Gymice with CaNT subcutaneous tumours were injected with a solution of thedrug at a dose of 16.7 μmol per kilogram. They were treated with theoptimum wavelength of light (determined in separated experiments) 1 hourafter injection. The light source was a Patterson lamp with appropriatefilters giving a bandwidth of 30 nm, and treatment was 60 J cm⁻² at arate of 50 mW cm⁻². It can be seen from FIG. 1 that tumour response isvery dependent on the nature of the alkyl groups, and the tetra-n-pentyland tetra-n-butyl derivatives were particularly effective compared withmethylene blue.

FIG. 2 shows the anti-tumour photodynamic efficacy (% tumour necrosis)for the tetra-n-propyl and tetra-n-pentyl derivatives as a function ofthe time interval between drug and light administration. These data showa quite unexpected difference between the two compounds. Thetetra-n-propyl derivative is very active at very short drug-light timeintervals (ie by giving light almost immediately after giving drug)whereas the tetra-n-pentyl derivative has very low activity at veryshort times, but much higher activity after 1 hour. The explanation forthis finding is as yet unknown, but clearly these differentialproperties could be exploited for different applications. For example,the fast acting drug could be used for vascular treatments and theslower acting drug could be used for tumour cell treatments.

The present compounds have a number of advantages over currentlyavailable compounds such as Photofrin (trade mark, Axcan Pharma PDT Inc)and Foscan (trade mark, Bioscience Technology Investment HoldingsLimited). For example the compounds of the present invention are singleisomer free compounds produced by relatively simple processes, whereasPhotofrin is a complex mixture of porphyrin derivatives. A short drugadministration to light interval is desirable both in terms of patientconvenience and time in hospital during treatment and associated costs.Photofrin requires a long drug administration to light interval,typically of 48 hours, otherwise unacceptable damage to normal tissuessurrounding the tumour occurs at short drug-to-light intervals. Thecompounds of the present invention, for example the tetra-n-pentylderivative provides a high level of tumour necrosis (70%) whereillumination is immediately after administration and 90% whereillumination is 1 hour after administration. To achieve comparablelevels of necrosis a 5-fold higher dose of Photofrin was required,

Comparison of damage to skin (assessed by scab formation) shows thatwith the present compounds, for example the tetra-n-pentyl derivative,no scab formation is observed at any drug to light interval whereasPhotofrin gave up to 25% scab formation at short drug to light intervals(0-3 hours), longer drug to light intervals of 48 hours gave no scabformation but tumour necrosis was only 50%.

For another available compound, Foscan, there is a delay of 4 days, toallow time for accumulation in the cancer cells, between injection intothe bloodstream and activation with laser light. Administration ofFoscan results in patients becoming highly sensitive to light, with athe period of sensitivity of approximately 15 days.

FIG. 3 shows the relative skin photosensitivity caused by thetetra-n-butyl and tetra-n-pentyl derivatives, compared withpolyhaematoporphyrin, PHP (equivalent to Photofrin). Photofrin is thecurrent leading PDT agent for oncology, but has the major disadvantageof causing prolonged skin photosensitivity in patients. In this model,the skin photosensitivity is measured in terms of the increase in earthickness 24 hours after exposure to drug and light. FIG. 3 shows that,as expected, PIHP shows a high level of skin photosensitivity, but thetwo phenothiazinium derivatives show little or no skin photosensitivity.These two derivatives also caused very little skin colouration based onadministration of the tetra-n-pentyl derivative to CBA/gy mice. Thedifference in skin coloration between pre- and post-drug administrationat dose rates of 16.7 μmol/kg, i.e 10 fold higher than necessary toachieve 90% tumour necrosis, does not induce any skin coloration.Coloration was comparable to control animals injected with saline.

Photo-Antimicrobial Activity

1) General Methods

Method for Microbial Bacterial Photoinactivation Experiment

a) Standard Preparation of Photosensitsiers

Stock solutions of the photosensitisers were made up to 5 mM indimethylsulfoxide (DMSO). The 5 mM stock was further diluted in DMSO toa working concentration of 1 mM. All photosensitisers were stored infoil covered vials at room temperature until required.

b) Standard Preparation of Microorganisms

The standard protocol outlined below was modified as appropriate tostudy variation of various experimental parameters.

A single bacterial colony from an agar plate was used to asepticallyinoculate 100 ml of nutrient media (0.5% yeast extract: 1.0% tryptonew/v) in a 1 I conical flask. For C. albicans a single fungal colony wasused to inoculate 100 ml of sabouraud dextrose media in a 1 l conicalflask. The culture was incubated in a shaking incubator overnight at 37°C. The incubator was set to 250 strokes per minute and a rotary motionof a 2.5 cm circle. This culture was used for the stationary phaseexperiments. For the log phase bacterial experiments the overnightculture was used to inoculate 200 ml of nutrient media (in a 2 lbevelled flask), for C. albicans 200 ml of sabouraud dextrose media wasinoculated, both were to an optical density of 0.1 at 600 nm. Themicroorganisms were grown until in the mid-logarithmic phase and thenharvested and resuspended.

c) Preparation of Microorganisms for PDT

The log or stationary phase cells were collected by centrifugation andwashed twice in 0.1 M potassium phosphate buffer (pH 7.0). Followingwashing, the cells were resuspended in the same buffer to an absorbanceof 0.87 at 650 nm. This absorbance was equivalent to 3.5×10⁸ CFU/ml or8.5 log₁₀CFU/ml for E. coli, S. aureus, MRSA and P. aeruginosa. For C.albicans this correlated to 1.0×10⁷ CFU/ml or 7.0 log₁₀CFU/ml. Forphotoinactivation of E. coli cells in media, the bacteria wereresuspended in nutrient media at this stage.

Microbial Cell Photoinactivation Experiments

Standard Incubation with Photosensitiser

25 ml of the prepared cell suspension was incubated with 0.25 mls of a 1mM stock solution of photosensitiser (giving a final concentration of 10μM) in a 250 ml sterile foil covered conical flask. The suspension wasincubated for 30 minutes in the dark in a 37° C. shaking incubator at250 rpm.

Illumination from a White Light Source

After incubation with 10 μM phenothiazinium compound, the suspension wasirradiated by a 500 W halogen lamp, from a distance of 75 cm, for 60minutes, the power of the lamp was 1.3 mW/cm² giving 4.68 J/cm² over thehour illumination.

Illumination from a 665 nm Laser

After incubation with 10 μM phenothiazinium compound, 10 ml of thebacterial culture was aseptically transferred to a sterile cell. Thisconsisted of a sealed vial with a sealed capillary tube inserted, intowhich the optical fibre could be placed. Illumination was carried outwith a Ceram Optec diode laser (665 nm) which used an optical fibre witha 3 cm diffusing tip, at 100 mW. For experiments comparing thephenothiazinium compound series in E. coli, samples were illuminated for4 min. This equated to a fluence rate of 5.3 mW/cm² assuming that thearea of the illuminated cylinder was 18.86 cm². After a 4 minillumination the total fluence was 1.3 J/cm². Other experiments used a10 min illumination and 50 μl samples were removed for CFU analysisafter 0, 1, 2, 4, 8 and 10 min illumination. These illumination timesare equivalent to the following fluences respectively: 0 J/cm²; 0.32J/cm²; 0.64 J/cm²; 1.3 J/cm²; 2.5 J/cm² or 3.2 J/cm². Oxygen electrodetraces showed oxygen was not limiting during the illumination period.For experiments comparing the effect oftetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound on differentbacteria illumination used the laser set up but for 10 minutes giving alight dose of 3.2 J/cm². This light dose was also used for experimentscomparing compounds 17-29. Results are tabulated above in Table 5 andbelow in Table 6.

Bacterial and Yeast Survival Analysis

50 ml of the illuminated and non-illuminated samples of the suspensionwere removed and diluted in 0.1 M pH 7.0 potassium phosphate buffer. 50μl of the diluted suspension was then plated on nutrient agar (0.5%yeast extract, 1.0% tryptone, 2.0% agar w/v) for bacteria, or sabourauddextrose agar for C. albicans. The plates were incubated overnight at37° C. to give a number of colony forming units between 30-300. Cellinactivation was then measured.

Control studies involving plating out of bacteria before and after the30 minute incubation step with no phenothiazinium compound but 0.25 mlsDMSO showed no change in CFU/ml. Illumination of the bacterial culturealone with no phenothiazinium compound but 0.25 mls DMSO alsodemonstrated no change in CFU. For illumination in nutrient media,control tests showed a log₁₀ increase in CFU/ml of 0.2 during the hourillumination.

Determination of the Effect of Phenothiazinium Compounds on BacterialCell Growth

200 ml of nutrient media (0.5% yeast extract 1.0% tryptone w/v) in foilcovered 250 ml conical flasks was aseptically innoculated with 10 ml ofa fully grown bacterial culture (E, coli). In addition the mediacontained 1.0 ml of a 1 mM stock solution of phenothiazinium compoundswith a final concentration of 10 μM, apart from the control whichcontained no phenothiazinium compounds but 1.0 ml DMSO.

The suspension was incubated at 37 C and 250 rpm in a shaking incubatorin the dark. 1 ml samples were taken every hour for 6 hours andturbidity based on apparent optical density at 550 nm caused by lightscattering was measured. Control studies show this wavelength is out ofthe region of photosensitiser absorption. Following optical densityreadings the 1.0 ml sample was spun in a MSE Micro-Centaur centrifuge(10 000 g×5 minutes) and the absorbance spectra of the supernatant readspectrophotometrically.

For the tetra-n-butyl derivative only, similar experiments were carriedout where the bacteria were allowed to grow without photosensitiser for3 hours, after which time the phenothiazinium compounds was added.Subsequent growth was monitored as a function of time, both for exposureto light and in the dark.

Uptake of the Photosensitisers into E. coli

Following incubation of the bacteria with photosensitiser, 2 ml of thenon-illuminated bacterial culture was sedimented using a BenchtopCentaur 5 centrifuge (1500 g×10 min). The bacterial pellet was washedtwice with 0.1 M potassium phosphate buffer (pH 7.0) to removeextracellular and loosely bound photosensitiser. Finally the pellet wasresuspended and vortexed in 1 ml of 0.1 M NaOH 2% (w/v) SDS and left atroom temperature, in the dark, for at least 24 h. Fluorescence readingswere taken using a Kontron SFM-25 spectrofluorimeter. The concentrationof phenothiazinium compound in the cellular samples was determined frominterpolation of the standard curves.

Photobleaching 0.25 mls of a 1 mM solution of photosensitiser, 0.25 mlsof 10 mM tryptophan was added to 25 mls of 60% methanol, 40% potassiumphosphate buffer (pH7.0). Experiments were also carried out in theabsence of tryptophan where this was replaced by 0.25 mls of the 60%methanol, 40% potassium phosphate buffer (pH7.0).

The mixture was illuminated as in the cell inactivation experimentsabove (1.3 mW/cm²) for 60 minutes, samples were taken every 15 minutesand spectra recorded on a UV-Visible spectrophotometer between 500 nmand 700 nm. For high light dose, illumination was at 9 mW/cm² for 60minutes.

Results

Antibacterial Properties of Phenothiazinium Derivatives

FIG. 4 shows log change in Colony Forming Units (CFU)/ml of E. coliincubated for 30 minutes with 10 μM phenothiazinium compound andilluminated for 60 minutes at 1.3 mW/cm². Data were recorded of cellsurvival following a 60 minute illumination by a halogen lamp. It may beseen that there is substantial bacterial inactivation with the trend inthe group being a decrease from methylene blue to ethylene blue,followed by an increase of almost 1000 fold up to the tetra-n-butylphenothiazinium derivative. The longer chain phenothiazinium compoundsthen show reduced bacterial cell kill ability such that thetetra-n-hexyl derivative is almost inactive. The tetra-n-butylphenothiazine led to the largest change in colony forming units per mlof 5.1 log₁₀ equivalent to a percentage cell survival of 0.001%. Thelowest change of 0.19 log₁₀CFU was using the tetra-n-hexyl derivativewhich is a cell survival of 65.3%. There was no cell inactivation with alight only control.

FIG. 5 shows the log change in CFU/ml of E. coli incubated for 30minutes with different concentrations of tetra-n-butyl phenothiaziniumderivative and illuminated for 15 minutes at 1.3 mW/cm⁻². 10 μM was themost effective concentration tested for bacterial inactivation usingtetra-n-butyl phenothiazinium derivative. The log change in CFU/ml withthis concentration was 4.59 log₁₀ units. Cell kill effects were achievedwith all of the concentrations tested but were reduced at the lower drugdoses. At 50 μM there is a log change of 2.15 units which is reducedcompared to 10 μM. This could be due to photosensitiser aggregation andtherefore lower drug doses to the cell.

Many antibiotics are only poorly effective against non-growing orstationary bacteria and it is important to assess the ability of thephenothiazinium compounds to inactivate stationary phase bacteria.During the stationary period the cell has a thicker peptidoglyan cellwall and differences in protein metabolism and therefore might be lesssusceptable to the photodynamic effect. FIG. 6 shows the log change inCFU/ml of E. coli in the stationary phase of growth following incubationfor 30 minutes with 10 μM phenothiazinium compounds and illuminated for60 minutes at 1.3 mW/cm². It may be seen that the effectiveness of thetetra-n-propyl and tetra-n-butyl derivatives is only slightly reducedagainst stationary phase bacteria, with again the tetra-n-butylderivative being the most effective.

Inactivation of bacteria may be more challenging in a therapeuticenvironment, because the sensitiser may bind preferentially toextracellular proteins rather than the bacterial lipopolysaccharidemembrane. This was tested by resuspending the bacteria in nutrientmedium containing many factors which might compete with bacterial cellsfor photosensitiser binding. FIG. 7 shows the log change in CFU/ml of E.coli resuspended in nutrient medium, from which it may be seen thatthere is little reduction in the level of cell kill. Again, thetetra-n-butyl phenothiazinium derivative has the highest antibacterialactivity.

FIG. 8 shows the log change in CFU/ml of E. coli following incubationwith 10 μM phenothiazinium compound for 30 minutes and illumination withlaser light (665 nm) for only 4 minutes at 0.1 W. Again, the samepattern of activity among the phenothiazine derivatives is seen, showingthat the effects are present with coherent laser light. The potentialadvantages of a laser source are increased accuracy of light doses andshorter illumination times.

Further studies with laser light showed that a log change of 5.69 CFU/mlcan be achieved with a 14 minute illumination at 0.1 W with thetetra-n-butyl phenothiazinium derivative and that after a 20 minuteillumination there is a log change of almost 8.5 units, though thenumber of CFU are too small to make this figure statistically reliable

Uptake of the photosensitisers into bacterial cells is clearly importantin determining photo-activity. FIG. 9 shows uptake of 10 μMphenothiazinium compounds into E. coli cells following a 30 minuteincubation. Cells were washed twice in 0.1M pH7.0 potassium phosphatebuffer to remove extra-cellular or loosely bound sensitiser. It may beseen that uptake of the phenothiazinium compounds is somewhat correlatedwith phototoxicity in that the tetra-n-butyl derivative is taken up themost by the bacterial cells. However, the correlation between uptake andphotoactivity is far from exact. For example, the ratio between thephotoactivity and the uptake for the tetra-n-butyl derivative is fargreater than that for the tetra-n-hexyl derivative. These ratios wouldbe expected to be the same for all of the derivatives if thephotoactivity could be explained only on the basis of uptake. It istherefore clear that the extremely high activities of the tetra-n-butyland tetra-n-propyl derivatives must be due to some additional factors,as yet unknown.

Data not shown have proved that the tetra-n-butyl derivative is taken upvery quickly into the E. coli; there are no differences in uptakebetween incubation times of 5 minutes and 30 minutes. However, in thepresence of nutrient medium, the uptake was found to be somewhat slowerand reduced in extent.

FIG. 10 shows the log change in CFU/ml of E. coli cells incubated with10 μM tetra-n-butyl phenothiazinium derivative and then washed twicewith 0.1M pH7 potassium phosphate buffer to remove any extra-cellular orloosely bound photosensitiser, which may have an effect on thephototoxicity. Illumination used laser light (665 nm) at 0.1 W for 4minutes. The results show there is little difference between the logchange in CFU/ml of washed and unwashed cells, indicating that it is thetightly bound photosensitiser which is causing cell death. At present,the precise location of the photosensitiser within the bacterial cell isnot known but the photodynamic action is effective and non-recovering.

Data for the effect of the different phenothiazinium compounds on thegrowth of an E. coli culture in the dark as compared to a control areshown in FIG. 11. Incubation was carried out in the dark at 37° C. for 6hours and measurements were based on apparent turbidity at 550 nm asdescribed earlier. All the cultures containing phenothiazinium compoundsshow reduced growth as compared to the control with the tetra-n-butylphenothiazinium derivative showing the greatest inhibition in the numberof cells in the bacterial suspension. It should be emphasised that thisdark inhibition is many orders of magnitude less than that observed forcell inactivation in the light.

Further work was carried out with the tetra-n-butyl derivative alone todetermine the effect of photosensitiser plus light on growth ofbacteria. These data, shown in FIG. 12 show clearly that for the growingbacteria with addition of photosensitiser after 3 hours, there iscontinuing growth in the dark, but complete elimination of growth in thelight. The data again illustrate the very powerful photobacteriocidaleffect of this photosensitiser.

FIG. 13 shows percentage cell survival of Pseudomonas aeruginosafollowing incubation with 10 μM tetra-n-butyl phenothiaziniumderivative. Illumination was with laser light (665 nm) at 0.1 W. Paeruginosa is a Gram negative organism which is commonly associated witha number of skin conditions including infections of ulcers and burnwounds. The figure shows that the tetra-n-butyl phenothiaziniumderivative can photodynamically inactive this organism extremelyefficiently. An illumination time of only 2 minutes with laser light(665 nm) at 0.1 W leads to a greater than 99% cell kill, while increaseof the illumination time to 10 minutes gives almost 4 logs of cell kill.Control studies showed that there is no reduction in cell number causedby the illumination alone in the absence of photosensitiser with 10 μMtetra-n-butyl phenothiazinium derivative.

FIG. 14 shows percentage cell survival of Staphylococcus aureusfollowing incubation with 10 μM tetra-nbutyl phenothiazinium.Illumination was with laser light (665 nm) at 0.1 W. S. aureus is a Grampositive organism which differs from Gram negative organisms in that ithas a thick outer peptidoglycan layer and no externallipopolysaccharide. The bacterial structure is the same as in MRSA(Methicillin resistant S. aureus) which is resistant to almost allcommonly used antibiotics. The data show that after only a 1 minuteillumination almost 99% of the bacteria are inactivated and that after10 minutes there is almost 5 logs of cell kill, illustrating the veryhigh photoactivity of the tetra-n-butyl derivative against this Grampositive organism.

It is important to determine if the photosensitiser would also be activeagainst the antibiotic resistant form, MRSA, as this would have majorhealth and industrial applications. FIG. 15 shows percentage cellsurvival of MRSA following illumination with 665 nm laser light at 0.1 Wand incubation with 10 μM tetra-n-butyl phenothiazinium derivative. Thedata clearly show that this photosensitiser is indeed highly photoactivein killing MRSA.

Anti-Fungal Properties of Phenothiazinium Derivatives

In order to test the ability of the tetrabutyl derivative to kill fungalcells in the light, the photosensitiser was incubated with cells ofCandida albicans and the culture was subjected to laser light asdescribed above. The results are shown in FIG. 16, in which it is clearthat this eukaryotic organism is also readily destroyed. Thisphotosensitiser is therefore also highly photoactive against this fungalorganism which is responsible for many common infections e.g. thrush.

Selectivity for Bacterial Cells Versus Mammalian Tissues

It is clearly important for therapeutic purposes that there is minimaldamage to host tissues while microorganisms are being destroyed. Thiswas tested by applying a solution of the tetra-n-butyl phenothiaziniumderivative to the ears of experimental mice and illuminating, underconditions in which the total dose was almost 20 times that needed forbacterial or fungal elimination. The possible effects on the host tissuewere assessed by measuring any increase in ear thickness. This is astandard model for detecting photodynamic reactions in the skin. FIG. 17shows the data obtained, compared with results from intravenousadministration of PHP, a drug equivalent to Photofrin which is known tocause prolonged skin reaction. It is clear from FIG. 17 that, while thereaction from PHP is very strong, as expected, there is little or noreaction from the tetra-n-butyl phenothiazinium derivative, suggestingthat mammalian tissues would not be damaged during antimicrobialtreatment.

Photobleaching

Photobleaching removes detectable colour from the photosensitiser,rendering it inactive and is the result of its instability to light andreduction or oxidation. Such photobleaching may have advantages ordisadvantages depending on the potential application. For example,photobleaching is undesirable in the coating of lines and catheters. Twosets of experiments were carried out; one at a high light dose (9.0mW/cm²) and one at a low light dose (1.3 mW/cm²) with and withouttryptophan as described above. Absorption spectra at low light dose,with and without tryptophan, showed no changes for any of thephenothiazinium compounds demonstrating they are stable at this dose. Atthe high light dose, spectral changes were observed for the methyleneblue, indicating photobleaching. The maximum absorbance decreased andthe wavelength peak shifted over the one hour illumination. Thesechanges occurred to the same extent with and without tryptophan.However, none of the other phenothiazinium compounds showed thisdegradation and remained stable to photobleaching at the high lightdose.

The antibacterial properties oftetra-n-pentyl-3,7-diaminophenothiazin-5-ium are tabulated below:

TABLE 6 Photoinactivation of bacteria and yeast in the log andstationary growth phase, following incubation with 10 μM photosensitiserand illumination with 665 nm laser light at a fluence rate of 3.2 J/cm².Cell kill (log Standard Photo- Growth reduction in error of sensitiserBacteria/Yeast phase CFU/ml) the mean Tetra-n- P. aeruginosa Log 3.880.27 pentyl-3,7- Stationary 1.28 0.22 diamino- S. aureus Log 4.21 0.22phenothi- Stationary 3.17 0.04 azin-5-ium MRSA Log 3.80 0.34 Stationary1.85 0.16 C. albicans Log 3.61 0.36

The data in the table above show the log reduction in CFU/ml of bacteriaor yeast incubated with 10 μM photosensitiser, and illuminated using a665 nm laser for 10 min. at a fluence of 3.2 J/cm².

The susceptibility of bacteria to phenothiazinium mediated PDT candepend on if the bacteria are Gram-positive or Gram-negative.Gram-positive bacteria (S. aureus, MRSA) have a highly cross linkedpeptidoglycan cell wall approximately 25 nm in thickness. Gram negativebacteria (E. coli, P. aeruginosa) have a thinner 5 nm cell wall and aunique lipopolysaccharide outer membrane. The presence of the outermembrane gives an increased resistance of Gram negative bacteria to manyantibacterial agents.

Following illumination the tetra-n-pentyl-3,7-diaminophenothiazin-5-iumcompound led to >3 log reduction in CFU/ml for both log phase, Gramnegative (E. coli, P. aeruginosa) and Gram positive bacteria (S. aureus,MRSA).

Many antibiotics have a low activity against bacteria in the stationarygrowth phase. Bacteria in the two growth phases differ in theirphysiology and morphology. Stationary phase cells are less active andmore resistant to environmental stress, therefore, may be resistant tophenothiazinium mediated PDT. The above table shows that theeffectiveness of the tetra-n-penty-3,7-diaminophenothiazin-5-iumcompound is only slightly reduced against stationary phase cellscompared to log phase cells.

MRSA, an antibiotic resistant strain of S. aureus is a major cause ofnosocomal infection. MRSA and S. aureus are equally susceptible totetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound mediated antimicrobial PDT. There was a log reduction of 3.80 log₁₀CFU/ml using thetetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound against log phaseMRSA.

Phenothiazinium Compounds of Formula I Suitable for Inclusion inPolymers or Attachment to, or Adsorption on, Polymer Surfaces

(a) Inclusion within Polymers

Example

To a clear solution of cellulose triacetate (0.5 g) in dichloromethane(10 cm³) was added sensitiser (Formula I in which A=B=NR¹R² andR¹=R²=n-Bu) (0.01 g) and the mixture was stirred until the sensitiserhad dissolved completely. The solution was then cast on a glass plateand allowed to dry slowly, giving a clear blue film. The film showedtypical singlet oxygen generating properties on exposure to light. Thusan aerated red solution of tetraphenylcyclopentadienone (acharacteristic singlet oxygen detector) in toluene containing the filmwas rapidly bleached on exposure to light from a 40 w tungsten filamentlamp. An identical solution showed no bleaching when irradiated for thesame period of time in the absence of the film.

(b) Adsorption on Polymers

Phenothiazinium compounds Ia and Ib were made according to the followingreaction scheme and were isolated as dark blue solids. They werecharacterised by mass spectrometry.

Compounds (Ia and Ib) were extremely basic and readily protonated indilute acids to give (IIa) and (IIb) respectively, which could beadsorbed strongly on polymeric surfaces, e.g. polyamides, polyacrylates,polyesters, polycarbonates, polyurethanes, and strongly resisted removalby water or solvents. Alternatively Ia or Ib could be adsorbed directlyonto acidic surfaces to give their corresponding cationic saltsdirectly.

(c) Covalent Attachment of the Phenothiazinium Sensitisers to PolymerSubstratesDerivatives Ia and Ib

Compounds Ia and Ib proved very reactive as nucleophiles in varioussubstitution reactions that could provide a means of attaching thesensitiser unit covalently to polymers.

Thus reaction with anhydrides occurred, as exemplified by the followingreaction:

Example:

Polyethylene-graft-maleic anhydride (1.0 g) was dissolved in toluene (25cm³) with warming. The sensitiser Ia (0.20 g) was added and reactionmixture heated under reflux for 1 hour. The mixture was poured intomethanol and the precipitate was filtered off, washed with methanol anddried, giving the sensitiser-bound copolymer as a dark blue powder (1.1g). Covalent attachment of the sensitiser to the polymer was confirmedby dissolving the powder in dichloromethane and precipitating it byaddition of methanol. No blue colour remained in the supernatant liquid.

A similar nucleophilic substitution reaction will occur with polymerscontaining ester

Phenothiazinium derivatives Ia and Ib are also very reactive towardschlorotriazine derivatives, and their linkage to polymers can be carriedout by the following procedure:

Where X=—NH-(Polymer) in the case of polyamide polymers

-   -   X=—O-(Polymer) in the case of cellulosic polymers

Alternatively, the residual chlorine in the previous example can bereplaced by other reactive groups, as in the following reaction:

Where X and Y=—NH-(polymer)Or X and Y=—O-(polymer)Or X may be an amine —NHR or —NRR′, and Y=—NH-(polymer), or —O-(polymer)

These are not the only means of attaching the phenothiaziniumderivatives to polymers, and other methods may by employed based onexisting polymer-grafting chemistry known to those skilled in the art.

Example:

Sodium carbonate (0.20 g) and cyanuric chloride (0.30 g) were added to asolution of the sensitiser Ia (0.26 g) in dry acetone (170 cm³) at roomtemperature, and the mixture was stirred for 15 minutes. A sheet oftransparent cellulose film (2.2 g) was immersed in an aqueous solutionof sodium hydroxide (1M; 500 cm³) for 10 minutes and then washed free ofsodium hydroxide. This was then introduced into the sensitiser solutionand the stirred mixture heated at 50° C. for 15 minutes. Water (200 ml)was added and the mixture heated at 60% for 30 minutes. The bluecellulose film was then removed and washed with water, and then heatedin sodium carbonate solution (6%) to remove any unfixed dye. Covalentattachment to the cellulose was confirmed by heating the film in boilingsodium carbonate solution or boiling methanol, when no blue colour wasremoved. The film showed typical singlet oxygen generating properties onexposure to light, and when immersed in an air-saturated solution oftetraphenylcyclopentadienone in toluene and exposed to light from a 40 wtungsten filament lamp, the red dienone was bleached more rapidly thanan identical solution containing no film.

REFERENCES

-   Wainwright M, Phoenix D A, Laycock S L, Wareing D R A, Wright P A.    (1998). Photobactericidal activity of phenothiazinium dyes against    methicillin-resistant strains of Staphylococcus aureus. FEMS    Microbiology Letters 160, 177-181.-   Wagner S J, Skripchenko A, Robinette D, Foley J W, Cincotta L    (1998). Factors affecting virus photoinactivation by a series of    phenothiazine dyes. Photochemistry and Photobiology 67, 343-349.

TABLE 2 Chemical properties of the phenothiazines and phototoxicity,dark toxicity, cellular uptake and subcellular localisation in RIF-1murine fibrosarcoma cells. Uptake at PDT Singlet oxygen PDT LD₅₀ Ratiodark: LD₅₀ (nmol/mg Initial Relocalisation + R generation¹ (μM)² PDTLD₅₀ ² protein)³ localisation⁴ light⁴ LogP Methyl 47 54 3 >4.6 LysosomesNucleus −1.0 Ethyl 42 3.9 27 2.9 Lysosomes Lysosomes +0.2 Propyl 40 0.4219 0.9 Lysosomes Mitochondria +1.1 Butyl 41 1.1 7 3.1 LysosomesMitochondria +1.1 Pentyl 39 0.74 10 1.6 Lysosomes Mitochondria +1.7Hexyl 35 2.1 4 1.6 Lysosomes Mitochondria +1.3 ¹% photo-oxidation of1,3-diphenylisobenzofuran after 10 minutes red light illumination with100 mg/ml of the phenothiazine in 90% DMF:10% water. ²Cells wereincubated with the phenothiazine for 2 h. For measurement ofphototoxicity, cells were illuminated with 3 J/cm² (10 mW/cm²) whitelight. Dark toxicity was measured in parallel. Cell survival wasassessed after 24 h using the MTT assay. ³Cells were incubated with thephenothiazine for 2 h. Cells were solubilised in 2% SDS and thephenothiazine levels measured by fluorescence. ⁴Cells were incubatedwith the PDT LD₅₀ concentration of the phenothiazine for 2 h andfluorescence images captured before and during 10 min illumination with630 nm light.

TABLE 3 Phototoxicity and dark toxicity of the phenothiazines in humantumour cell lines. R OE33¹ SiHa² HT1376³ HT29⁴ Methyl PDT LD₅₀ 43.5 ±1.8  18.7 ± 1.0  37.9 ± 10.1 88.5 ± 6.1  (μM) Ratio dark: 2.0 1.0 1.61.5 PDT LD₅₀ Propyl PDT LD₅₀ 0.30 ± 0.09 0.075 ± 0.015 0.20 ± 0.13 0.22± 0.04 (μM) Ratio dark: 11 80 13 18 PDT LD₅₀ Butyl PDT LD₅₀ 0.28 ± 0.06(μM) Ratio dark: 18 PDT LD₅₀ Pentyl PDT LD₅₀ 0.29 ± 0.06 0.75 ± 0.221.49 ± 0.41 (μM) Ratio dark: 11 6 4 PDT LD₅₀ ¹oesophageal adenocarcinoma²cervical squamous cell carcinoma ³bladder transitional cell carcinoma⁴colon adenocarcinoma Cells were incubated with the phenothiazine for 2h. For measurement of phototoxicity, cells were illuminated with 3 J/cm²(10 mW/cm²) 665 nm light. Dark toxicity was measured in parallel. Cellsurvival was assessed after 48 h using the sulforhodamine B (SRB) assay.

1. A pharmaceutical composition which contains a compound of formula

in which R¹, R², R³ and R⁴ are each n-butyl, where X^(P−) is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant, which composition includes liposomes, nano-particles, colloidal suspensions, micelles, micro-emulsions, vesicles or nano-spheres.
 2. The composition according to claim 1, which composition includes liposomes.
 3. A pharmaceutical composition which contains a compound of formula

in which R¹, R², R³ and R⁴ are each n-butyl, where X^(P−) is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant, which composition includes a delivery vehicle or excipient comprising an isotonising agent selected from the group consisting of urea, glycerol, aminoethanol and propylene glycol.
 4. The composition according to claim 3, wherein said isotonising agent comprises propylene glycol.
 5. A pharmaceutical composition which contains a compound of formula

in which R¹, R², R³ and R⁴ are each n-butyl, where X^(P−) is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant, wherein the concentration of said compound is in the range 100 μM to 30 mM.
 6. A pharmaceutical composition which contains a compound of formula

in which R¹, R², R³ and R⁴ are each n-butyl, where X^(P−) is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant, wherein said compound includes Br⁻ as a counterion.
 7. A method of preparing a composition according to claim 1, the method comprising mixing said compound with one or more pharmaceutically acceptable carriers.
 8. A method according to claim 7 comprising mixing said compound with said one or more pharmaceutically acceptable carriers at a pH of 3 to
 9. 9. A method according to claim 7 comprising mixing said compound with said one or more pharmaceutically acceptable carriers at a pH of 6.5 to 7.5.
 10. A pharmaceutical composition which contains a compound of formula

in which R¹, R², R³ and R⁴ are each n-butyl, where X^(P−) is a counteranion and P is 1, 2 or 3, together with a pharmaceutically acceptable carrier, excipient or adjuvant, which composition also includes a compound of formula

wherein: A and B each independently is

in which R′ and R″ each independently is an optionally substituted linear, branched or cyclic hydrocarbon group, or R′ and R″ together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7-membered ring; X^(P−) is a counteranion and P is 1, 2 or 3; or one or more other different therapeutic or active agents. 